This invention relates to peptides which are fragments of protein products arising from frameshift mutations in genes, which peptides elicit T cellular immunity, and to cancer vaccines and compositions for anticancer treatment comprising said peptides.
The invention further relates to a method for identifying such peptides which are fragments of protein products arising from frameshift mutations in genes, which may elicit T cellular immunity which is useful for combating cancer associated with said mutated genes.
The invention also relates to DNA sequences encoding at least one frameshift mutant peptide, and vectors comprising at least one insertion site containing a DNA sequence encoding at least one frameshift mutant peptide.
Further the invention relates to methods for the treatment or prophylaxis of cancers associated with frameshift mutations in genes by administration of at least one frameshift mutant peptide or a recombinant virus vector comprising at least one insertion site containing a DNA sequence encoding at least one frameshift mutant peptide, or an isolated DNA sequence comprising a DNA sequence encoding at least one frameshift mutant peptide.
The present invention represents a further development of anticancer treatment or prophylaxis based on the use of peptides to generate activation and strengthening of the anti cancer activity of the T cellular arm of the body""s own immune system.
Tumour Antigens, Status:
T cell defined antigens have now been characterised in a broad spectrum of cancer types. These antigens can be divided into several main groups, depending on their expression. The two main groups are constituted by developmental differentiation related antigens (tumour-testis antigens, oncofoetal antigens etc., such as MAGE antigens and CEA) and tissue specific differentiation antigens (Tyrosinase, gp100 etc.). The group containing the truly tumour specific antigens contains proteins that are altered due to mutations in the genes encoding them. In the majority of these, the mutations are unique and have been detected in a single or in a small number of tumours. Several of these antigens seem to play a role in oncogenesis.
Cancer Vaccines, Status:
The focus in cancer vaccine development has been on antigens expressed in a high degree within one form of cancer (such as melanoma) or in many kinds of cancers. One reason for this is the increased recruitment of patients into clinical protocols. The field is in rapid growth, illustrated by the accompanying table listing the cancer vaccine protocols currently registered in the PDQ database of NCI.
Inheritable Cancer/Cancer Gene Testing:
Inherited forms of cancer occur at a certain frequency in the population. For several of these cancer forms, the underlying genetic defects have been mapped. This is also the case in Lynch syndrome cancers which constitute an important group of inheritable cancer. In families inflicted with this syndrome, family members inherit defect genes encoding DNA Mismatch Repair (MMR) Enzymes. Carriers of such MMR defects frequently develop colorectal cancer (HNPCC) and other forms of cancer (list?). Mutations in MMR enzymes can be detected using gene testing in the same way as other cancer related genes can be detected.
Gene testing of risk groups in this case represents an ethical dilemma, since no acceptable forms for prophylactic treatment exist. At present surgery to remove the organ in danger to develop cancer has been the only treatment option. In these patients, cancer vaccines will be a very (interesting) form of prophylaxis, provided efficient vaccines can be developed.
The lack of efficient repair of mismatched DNA results in deletions and insertions in one strand of DNA, and this happens preferentially in stretches of DNA containing repeated units (repeat sequences). Until now, focus has been on repeat sequences in the form of non-coding microsattelite loci. Indeed microsattelite instability is the hallmark of cancers resulting from MMR defects. We have taken another approach, and have concentrated on frameshift mutations occurring in DNA sequences coding for proteins related to the oncogenic process. Such frameshift mutations result in completely new amino acid sequences in the C-terminal part of the proteins, prematurely terminating where a novel stop codon appears. This results in two important consequences:
1) The truncated protein resulting from the frameshift is generally nonfunctional, in most cases resulting in xe2x80x9cknocking outxe2x80x9d of an important cellular function. Aberrant proteins may also gain new functions such as the capacity to aggragate and form plaques. In both cases the frameshift results in disease.
2) The short new C-terminal amino acid sequence resulting from the shift in the reading frame (the xe2x80x9cframeshift sequencexe2x80x9d) is foreign to the body. It does not exist prior to the mutation, and it only exists in cells having the mutation, i.e. in tumour cells and their pre malignant progenitors. Since they are completely novel and therefore foreign to the immune system of the carrier, they may be recognised by T-cells in the repertoire of the carrier. So far, nobody has focused on this aspect of frameshift mutations, and no reports exist on the characterisation of frameshift peptides from coding regions of proteins as tumour antigens. This concept is therefore novel and forms the basis for developing vaccines based on these sequences. It follows that such vaccines may also be used prophyllactively in persons who inherit defective enzymes belonging to the MMR machinery. Such vaccines will therefore fill an empty space in the therapeutic armament against inherited forms of cancer.
It has been shown that single amino acid substitutions in intracellular xe2x80x9cselfxe2x80x9d-proteins may give rise to tumour rejection antigens, consisting of peptides differing in their amino acid sequence from the normal peptide. The T cells which recognise these peptides in the context of the major histocompatibility (MHC) molecules on the surface of the tumour cells, are capable of killing the tumour cells and thus rejecting the tumour from the host.
In contrast to antibodies produced by the B cells, which typically recognise a free antigen in its native conformation and further potentially recognise almost any site exposed on the antigen surface, T cells recognise an antigen only if the antigen is bound and presented by a MHC molecule. Usually this binding will take place only after appropriate antigen processing, which comprises a proteolytic fragmentation of the protein, so that the resulting peptide fragment fits into the groove of the MHC molecule. Thereby T cells are enabled to also recognise peptides derived from intracellular proteins. T cells can thus recognise aberrant peptides derived from anywhere in the tumour cell, in the context of MHC molecules on the surface of the tumour cell, and can subsequently be activated to eliminate the tumour cell harbouring the aberrant peptide.
M. Barinaga, Science, 257, 880-881, 1992 offers a short review of how MHC binds peptides. A more comprehensive explanation of the Technical Background for this Invention may be found in D. Male et al, Advanced Immunology, 1987, J.B.lippincott Company, Philadelphia. Both references are hereby included in their entirety.
The MHC molecules in humans are normally referred to as HLA (human leukocyte antigen) molecules. They are encoded by the HLA region on the human chromosome No 6.
The HLA molecules appear as two distinct classes depending on which region of the chromosome they are encoded by and which T cell subpopulations they interact with and thereby activate primarily. The class I molecules are encoded by the HLA A, B and C subloci and they primarily activate CD8+ cytotoxic T cells. The HLA class II molecules are encoded by the DR, DP and DQ subloci and primarily activate CD4+ T cells, both helper cells and cytotoxic cells.
Normally every individual has six HLA Class I molecules, usually two from each of the three groups A, B and C. Correspondingly, all individuals have their own selection of HLA Class II molecules, again two from each of the three groups DP, DQ and DR. Each of the groups A, B, C and DP, DQ and DR are again divided into several subgroups. In some cases the number of different HLA Class I or II molecules is reduced due to the overlap of two HLA subgroups.
All the gene products are highly polymorphic. Different individuals thus express distinct HLA molecules that differ from those of other individuals. This is the basis for the difficulties in finding HLA matched organ donors in transplantations. The significance of the genetic variation of the HLA molecules in immunobiology is reflected by their role as immune-response genes. Through their peptide binding capacity, the presence or absence of certain HLA molecules governs the capacity of an individual to respond to peptide epitopes. As a consequence, HLA molecules determine resistance or susceptibility to disease.
T cells may control the development and growth of cancer by a variety of mechanisms. Cytotoxic T cells, both HLA class I restricted CD8+ and HLA Class II restricted CD4+, may directly kill tumour cells carrying the appropriate tumour antigens. CD4+ helper T cells are needed for cytotoxic CD8+ T cell responses as well as for antibody responses, and for inducing macrophage and LAK cell killing.
A requirement for both HLA class I and II binding is that the peptides must contain a binding motif, which usually is different for different HLA groups and subgroups. A binding motif is characterised by the requirement for amino acids of a certain type, for instance the ones carrying large and hydrophobic or positively charged side groups, in definite positions of the peptide so that a narrow fit with the pockets of the HLA binding groove is achieved. The result of this, taken together with the peptide length restriction of 8-10 amino acids within the binding groove, is that it is quite unlikely that a peptide binding to one type of HLA class I molecules will also bind to another type. Thus, for example, it may very well be that the peptide binding motif for the HLA-A1 and HLA-A2 subgroups, which both belong to the class I gender, are as different as the motifs for the HLA-A1 and HLA-B1 molecules.
For the same reasons it is not likely that exactly the same sequence of amino acids will be located in the binding groove of the different class II molecules. In the case of HLA class II molecules the binding sequences of peptides may be longer, and it has been found that they usually contain from 10 to 16 amino acids, some of which, at one or both terminals, are not a part of the binding motif for the HLA groove.
However, an overlap of the different peptide binding motifs of several HLA class I and class II molecules may occur. Peptides that have an overlap in the binding sequences for at least two different HLA molecules are said to contain xe2x80x9cnested T cell epitopesxe2x80x9d. The various epitopes contained in a xe2x80x9cnested epitope peptidexe2x80x9d may be formed by processing of the peptide by antigen presenting cells and thereafter be presented to T cells bound to different HLA molecules. The individual variety of HLA molecules in humans makes peptides containing nested epitopes more useful as general vaccines than peptides that are only capable of binding to one type of HLA molecule.
Effective vaccination of an individual can only be achieved if at least one type of HLA class I and/or II molecule in the patient can bind a vaccine peptide either in it""s full length or as processed and trimmed by the patient""s own antigen presenting cells.
The usefulness of a peptide as a general vaccine for the majority of the population increases with the number of different HLA molecules it can bind to, either in its full length or after processing by antigen presenting cells.
In order to use peptides derived from a protein encoded by a mutated gene as vaccines or anticancer agents to generate anti tumour CD4+ and/or CD8+ T cells, it is necessary to investigate the mutant protein in question and identify peptides that are capable, eventually after processing to shorter peptides by the antigene presenting cells, to stimulate T cells.
In our International Application PCT/NO92/00032 (published as WO92/14756), the content of which is herein incorporated by reference we described synthetic peptides and fragments of oncogene protein products which have a point of mutation or translocations as compared to their proto-oncogene or tumour suppressor gene protein. These peptides correspond to, completely cover or are fragments of the processed oncogene protein fragment or tumour suppressor gene fragment as presented by cancer cells or other antigen presenting cells, and are presented as a HLA-peptide complex by at least one allele in every individual. These peptides were also shown to induce specific T cell responses to the actual oncogene protein fragment produced by the cell by processing and presented in the HLA molecule. In particular, we described peptides derived from the p21 ras protein which had point mutations at particular amino acid positions, namely position 12, 13 and 61. These peptides have been shown to be effective in regulating the growth of cancer cells in vitro. Furthermore, the peptides were shown to elicit CD4+ T cell immunity against cancer cells harbouring the mutated p21 ras oncogene protein through the administration of such peptides in vaccination or cancer therapy schemes. Later we have shown that these peptides also elicit CD8+ T cell immunity against cancer cells harbouring the mutated p21 ras oncogene protein through the administration mentioned above.
However, the peptides described above will be useful only in certain number of cancers, namely those which involve oncogenes with point mutations or translocation in a proto-oncogene or tumour suppressor gene. There is therefore a strong need for an anticancer treatment or vaccine which will be effective against a more general range of cancers.
In general, tumors are very heterogenous with respect to genetic alterations found in the tumour cells. This implies that both the potential therapeutic effect and prophylactic strength of a cancer vaccine will increase with the number of targets that the vaccine is able to elicit T cell immunity against. A multiple target vaccine will also reduce the risk of new tumour formation by treatment escape variants from the primary tumour.
There is a continuing need for new anticancer agents based on antigenic peptides giving rise to specific T cellular responses and toxicity against tumours and cancer cells carrying genes with mutations related to cancer. The present invention will contribute largely to supply new peptides that can have a use in the combat and prevention of cancer as ingredients in a multiple target anti-cancer vaccine.
Another problem solved by the present invention is that a protection or treatment can be offered to the individuals belonging to family""s or groups with high risk for hereditary cancers. Hereditary cancers are in many cases associated with genes susceptible to frameshift mutations as described in this invention (i.e. mutations in mismatch repair genes). Today it is possible to diagnose risk of getting hereditary cancer but no pharmaceutical method for protection against the onset of the cancer is available.
A main object of the invention is to obtain peptides corresponding to peptide fragments of mutant proteins produced by cancer cells which can be used to stimulate T cells.
Another main object of the invention is to develop a cancer therapy for cancers based on the T cell immunity which may be induced in patients by stimulating their T cells either in vivo or in vitro with the peptides according to the invention.
A third main object of the invention is to develop a vaccine to prevent the establishment of or to eradicate cancers based solely or partly on peptides corresponding to peptides of the present invention which can be used to generate and activate T cells which produce cytotoxic T cell immunity against cells harbouring the mutated genes.
A fourth main object of the invention is to design an anticancer treatment or prophylaxis specifically adapted to a human individual in need of such treatment or prophylaxis, which comprises administering at least one peptide according to this invention.
These and other objects of the invention are achieved by the attached claims.
Since frameshift mutations result in premature stop codons and therefore deletion in large parts of the proteins, proteins with frameshift mutations have generally not been considered to be immunogenetic and have therefore not been considered as targets for immunotherapy. Thus it has now surprisingly been found that a whole group of new peptides resulting from frameshift mutations in tumour relevant genes are useful for eliciting T cell responses against cancer cells harbouring genes with such frameshift mutations.
Genes containing a mono nucleoside base repeat sequence of at least five residues, for example of eighth deoxyadenosine bases (AAAAAAAA), or a di-nucleoside base repeat sequence of at least four di-nucleoside base units, for example of two deoxyadenosine-deoxycytosine units (ACAC), are susceptible to frameshift mutations. The frameshift mutations occur, respectively, either by insertion of one or two of the mono-nucleoside base residue or of one or two of the di-nucleoside base unit in the repeat sequence, or by deletion of one or two of the mono-nucleoside base residue or of one or two of the di-nucleoside base unit from the repeat sequence. A gene with a frameshift mutation will from the point of mutation code for a protein with a new and totally different amino acid sequence as compared to the normal gene product. This mutant protein with the new amino acid sequence at the carboxy end will be specific for all cells carrying the modified gene.
In the remainder of this specification and claims the denomination frameshift mutant peptides will comprise such proteins and peptide fragments thereof.
It has now according to the present invention been found that such new protein sequences arising from frameshift mutations in genes in cancer cells give rise to tumour rejection antigens that are recognised by T cells in the context of HLA molecules.
It has further according to the present invention been found a group of peptides corresponding to fragments of mutant proteins arising from frameshift mutations in genes in cancer cells which can be used to generate T cells. The said peptides can therefore also be used to rise a T cell activation against cancer cells harbouring a gene with a frameshift mutation as described above.
These peptides are at least 8 amino acids long and correspond, either in their full length or after processing by antigen presenting cells, to the mutant gene products or fragments thereof produced by cancer cells in a human patient afflicted with cancer.
A peptide according to this invention is characterised in that it
a) is at least 8 amino acids long and is a fragment of a mutant protein arising from a frameshift mutation in a gene of a cancer cell; and
b) consists of at least one amino acid of the mutant part of a protein sequence encoded by said gene; and
c) comprises 0-10 amino acids from the carboxyl terminus of the normal part of the protein sequence preceding the amino terminus of the mutant sequence and may further extend to the carboxyl terminus of the mutant part of the protein as determined by a new stop codon generated by the frameshift mutation in the gene; and
d) induces, either in its full length or after processing by antigen presenting cell, T cell responses.
The peptides of this invention contain preferably 8-25, 9-20, 9-16, 8-12 or 20-25 amino acids. They may for instance contain 9, 12, 13, 16 or 21 amino acids.
It is most preferred that the peptides of the present invention are at least 9 amino acids long, for instance 9-18 amino acids long, but due to the processing possibility of the antigen presenting cells also longer peptides are very suitable for the present invention. Thus the whole mutant amino acid sequence may be used as a frameshift mutant peptide according to the present invention, if it comprises 8 amino acids or more.
The invention further relates to a method for vaccination of a person disposed for cancer, associated with a frameshift mutation in a gene, consisting of administering at least one peptide of the invention one or more times in an amount sufficient for induction of T-cell immunity to the mutant proteins encoded by the frameshift mutated gene.
The invention also relates to a method for treatment of a patient afflicted with cancer associated with frameshift mutation in genes, consisting of administering at least one peptide of the invention one or more times in an amount sufficient for induction of T-cell immunity to mutant proteins arising from frameshift mutations in the genes of cancer cells.
Furthermore, it has according to the present invention been found a method for identifying new peptides which correspond to fragments of proteins arising from frameshift mutations in genes. This method is characterised by the following steps:
1) identifying a gene in a cancer cell susceptible to frameshift mutation by having a mono nucleoside base repeat sequence of at least five residues, or a di-nucleoside base repeat sequence of at least four di-nucleoside base units; and
2) removing, respectively, one nucleoside base residue or one di-nucleoside base unit from the repeat sequence and identifying the amino acid sequence of the protein encoded by the altered gene sequence as far as to include a new stop codon; and/or
3) removing, respectively, two nucleoside base residues or two di-nucleoside base units from the repeat sequence and identifying the amino acid sequence of the protein encoded by the altered gene sequence as far as to include a new stop codon; and/or
4) inserting, respectively, one nucleoside base residue or one di-nucleoside base unit in the repeat sequence and identifying the amino acid sequence of the protein encoded by the altered gene sequence as far as to include a new stop codon; and/or
5) inserting, respectively, two nucleoside base residues or two di-nucleoside base units in the repeat sequence and identifying the amino acid sequence of the protein encoded by the altered gene sequence as far as to include a new stop codon.
In order to determine whether the peptides thus identified are useable in the compositions and methods according to the present invention for the treatment or prophylaxis of cancer, the following further step should be performed:
6) determining whether the new peptide, either in their full length or as shorter fragments of the peptides, are able to stimulate T cells.
Optionally a further step may be added as follows:
7) determining peptides containing nested epitopes for different major HLA class I and/or HLA class II molecules.
In the present description and claims, the amino acids are represented by their one letter abbreviation as known in the art.
The peptides of the present invention shall be explicitly exemplified through two different embodiments, wherein cancer develops based on frameshift mutations in specific genes, namely the BAX gene and TGFxcex2RII gene:
I) BAX Gene
It has been established that the BAX gene is involved in regulation of survival or death of cells by promoting apoptosis. The human BAX gene contains a repeat sequence of eight deoxyguanosine bases (G8) in the third exon, spanning codons 38 to 41 (ATG GGG GGG GAG).
Frameshift mutations in this G8 repeat have been observed, both as G7 (ATG GGG GGG AGG) and G9 (ATG GGG GGG GGA) repeats, both in colon cancer cells and prostate cancer cells. The occurency is more than 50% of the examined cases (Rampino, N. et al., xe2x80x9cSomatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype.xe2x80x9d, Science (Washington D.C.), 275: 967-969, 1997). The modified BAX gene products are unable to promote apoptosis and thus makes further tumour progress possible. Furthermore the modified gene products are only found in cancer cells and are therefore targets for specific immunotherapy.
According to the present invention, peptides corresponding to the transformed BAX protein products arising from frameshift mutations in the BAX gene can be used as anticancer therapeutical agents or vaccines with the function to trigger the cellular arm of the immune system (T-cells) against cancer cells in patients afflicted with cancers associated with a modified BAX gene.
Frameshift mutations in the BAX gene result in mutant peptide sequences with the first amino acid of the altered sequence in position 41 as compared to the normal BAX protein (Table 1, seq.id. no. 1 to 4).
Table 2 shows one group of peptides according to the present invention:
The peptides listed in Table 3 were used for in vitro generation of T cells that recognise mutant BAX peptides.
The most preferred peptides according to this embodiment of the present invention are listed in Table 4:
2) TGFxcex2RII
It has been established that the TGFxcex2RII gene is involved in regulation of cell growth. TGFxcex2RII is a receptor for TGFxcex2 which down regulates cell growth. The human gene coding for TGFxcex2RII contains a repeat sequence of ten deoxyadenosine bases (A10) from base no. 709 to base no. 718 (GAA AAA AAA AAG CCT). In colon cancers and pancreatic cancers frameshift mutations in this A10 repeat have been observed, both as A9 (GAA AAA AAA AGC CT) and A11 (GAA AAA AAA AAA GCC) repeats, in approximately 80% of the examined cases (Yamamoto, H., xe2x80x9cSomatic frameshift mutations in DNA mismatch repair and proapoptosis genes in hereditary nonpolyposis colorectal cancer.xe2x80x9d, Cancer Research 58, 997-1003, Mar. 1, 1998). The modified TGFxcex2RII gene products are unable to bind TGFxcex2 and the signal for down regulation of cell growth is eliminated and thus makes further tumour progress possible. Furthermore the modified gene products are only found in cancer cells and are therefore targets for immunotherapy.
Consequently peptides corresponding to the transformed TGFxcex2RII protein products arising from frameshift mutations in the TGFxcex2RII gene can be used as anticancer therapeutical agents or vaccines with the function to trigger the cellular arm of the immune system (T-cells) against cancer cells in patients afflicted with cancers associated with a modified TGFxcex2RII gene.
Frameshift mutations in the TGFxcex2RII gene result in mutant peptide sequences with the first amino acid of the altered sequence in either position 133 (one and two base deletions) or 134 (one and two base insertions) as compared to the normal TGFxcex2RII protein (Table 5, seq.id.nos. 13 and 21).
Table 6 shows one groups of peptides of this invention:
Table 7 presents peptides that were used for in vitro generation of T cells that recognise mutant TGFxcex2RII peptides.
The most preferred peptides of this embodiment of the present invention are:
Other peptides of the invention can be fragments of the peptides listed in the Tables 1-8 above. Such fragments are most preferred from 9-16 amino acids long and include at least one amino acid from the mutant part of the protein.
As used in this description and claims the term fragment is intended to specify a shorter part of a longer peptide or of a protein.
Other cancer associated genes containing repeat sequences of a nucleoside base and which therefore are susceptible to frameshift mutations and consequently are potential candidates for peptides according to the present invention (seq id nos according to table 9 are given in parentheses in each case) are the following:
Human TGF-xcex2-2 (hTGFxcex22) gene (seq id nos 22-29)
Deleted in colorectal cancer (DCC) gene (seq.id.nos. 30-34)
Human breast and ovarian cancer susceptibility (BRCA1) gene (seq.id.nos. 378-387)
Human breast cancer susceptibility (BRCA2) gene (seq.id.nos. 35-94)
Human protein tyrosine phosphatase (hPTP) gene (seq.id.nos. 95-102)
Human DNA topoisomerase II (top2) gene (seq.id.nos. 103-108)
Human kinase (TTK) gene (seq.id.nos. 109-120)
Human transcriptional repressor (CTCF) gene (seq.id.nos. 121-127)
Human FADD-homologous ICE/CED-3-like protease gene (seq.id.nos. 128-133)
Human putative mismatch repair/binding protein (hMSH3) gene (seq.id.nos. 134-147)
Human retinoblastoma binding protein 1 isoform I (hRBP1) gene (seq.id.nos. 148-156)
Human FMR1 (hFMR1) gene (seq.id.nos. 157-161)
Human TINUR gene (seq.id.nos. 162-169) b-raf oncogene (seq.id.nos. 170-175)
Human neurofibromin (NF1) gene (seq.id.nos. 176-181)
Human germline n-myc gene (seq.id.nos. 182-188)
Human n-myc gene (seq.id.nos. 189-194)
Human ras inhibitor gene (seq.id.nos. 195-199)
Human hMSH6 gene (seq.id.nos. 200-203 and 293-297)
Human nasopharynx carcinoma EBV BNLF-1 gene (seq.id.nos. 204-210)
Human cell cycle regulatory protein (E1A-binding protein) p300 gene (seq.id.nos. 211-218)
Human B-cell lymphoma 3-encoded protein (bcl-3) gene (seq.id.nos. 219-226)
Human transforming growth factor-beta induced gene product (BIGH3) (seq.id.nos. 227-232)
Human transcription factor ETV1 gene (seq.id.nos. 233-239)
Human insulin-like growth factor binding protein (IGFBP4) gene (seq.id.nos. 240-246)
Human MUC1 gene (seq.id.nos. 247-266)
Human protein-tyrosine kinase (JAK1) gene (seq.id.nos. 267-271)
Human protein-tyrosine kinase (JAK3) gene (seq.id.nos. 272-279)
Human Flt4 gene (for transmembrane tyrosinase kinase) (seq.id.nos. 280-284)
Human p53 associated gene (seq.id.nos. 285-292)
Human can (hCAN) gene (seq.id.nos. 298-300)
Human DBL (hDBL) proto-oncogene/Human MCF2PO (hMCF2PO) gene (seq.id.nos. 301-306)
Human dek (hDEK) gene (seq.id.nos. 307-309)
Human retinoblastoma related protein (p107) gene (seq.id.nos. 310-313)
Human G protein-coupled receptor (hGPR1) gene (seq.id.nos. 314-319)
Human putative RNA binding protein (hRBP56) gene (seq.id.nos. 320-325)
Human transcription factor (hITF-2) gene (seq.id.nos. 326-327)
Human malignant melanoma metastasis-supressor (hKiSS-1) gene (seq.id.nos. 328-334)
Human telomerase-associated protein TP-1 (hTP-1) gene (seq.id.nos. 335-348)
Human FDF-5 (hFDF-5) gene (seq.id.nos. 349-356)
Human metastasis-associated mta1 (hMTA1) gene (seq.id.nos. 357-362)
Human transcription factor TFIIB 90 kDa subunit (hTFIIB90) gene (seq id nos 363-369)
Human tumour suppressor (hLUCA-1) gene (seq id nos 370-377)
Human Wilm""s tumour (WIT-1) associated protein (seq id nos 388-393)
Human cysteine protease (ICErel-III) gene (seq id nos 394-398 and 459)
Human Fas ligand (FasL) gene (seq id nos 399-403)
Human BRCA1-associated RING domain protein (BARD1) gene (seq id nos 404-417)
Human mcf.2 (hMCF.2) gene (seq id nos 418-422)
Human Fas antigen (fas) gene (seq id nos 423-427)
Human DPC4 gene (seq id nos 429-437).
The mutant peptides that are the results of frameshift mutation in these genes, in accordance with the present invention, are listed in table 9.
Examples of cancers particularly suitable for treatment with one or a combination of several of this compounds are: colorectal cancer, breast cancer, small-cell lung cancer, non small-cell lung cancer, liver cancer (primary and secondary), renal cancer, melanoma, ovarian cancer, cancer of the brain, head and neck cancer, pancreatic cancer, gastric cancer, eosophageal cancer, prostate cancer and leukemias and lymphomas.
Below are listed some examples of where these mutations may result in gene products that result in development of tumours:
Development of colorectal cancers are believed to result from a series of genetic alterations. Deleted in colorectal cancer (DCC) gene (seq id nos 30-34), Human cysteine protease (ICErel-III) gene (seq id nos 394-398 and 459), Human putative mismatch repair/binding protein (hMSH3) gene (Seq id nos 134-147), Human hMSH6 gene (seq id nos 200-203 and 293-297), Human n-myc gene (seq id nos 189-194), Human TGFxcex22 (hTGFxcex22) gene (seq id nos 22-29), Human p53 associated gene (seq id nos 285-292) may be involved in colorectal cancer.
Human breast cancer susceptibility (BRCA2) (seq id nos 35-94) and Human BRCA1-associated RING domain protein (BARD1) gene (seq id nos 404-417) are involved in breast cancer and ovarian cancer Human hMSH6 gene (seq id nos 200-203 and 293-297) may be involved in brain tumours.
Gene alteration are frequent in many types of adenocarcinomas, below are listed some genes that are mutated in many cancers:
Human breast cancer susceptibility (BRCA2) gene (seq id nos 35-94), Deleted in colorectal cancer (DCC) gene (seq id nos 30-34), Human putatative mismatch repair/binding protein (hMSH3) gene (seq id nos 134-147), Human hMSH6 gene (seq id nos 200-203 and 293-297), human N-MYC gene (seq id no 189-194), Human TGFb2 (hTGFb2) gene (seq id nos 22-29), Human p53 associated gene (seq id nos 285-292), Human MUC1 gene (seq id nos 247-266), Human germline n-myc gene (seq id nos 182-188), Human Wilm""s tumour (WIT-1) associated protein (seq id nos 388-393), Human nasopharynx carcinoma EBV BNLF-1 gene (seq id nos 204-210), Human transforming growth factor-beta induced gene product (BIGH3) seq id nos 227-232).
Many of the mutated genes may result in development of leukemias and lymphomas: Human neurofibromin (NF1) gene (seq id nos 176-181), b-raf oncogene (seq id nos 170-175), Human protein-tyrosine kinase (JAK1) gene (seq id nos 267-271), Human protein-tyrosine kinase (JAK3) gene (seq id nos 272-279) are examples.
Genes involved in malignant melanoma: Human malignant melanoma (metastasis-supressor (hKiSS-1) gene (seq id nos 328-334), Genes involved in metastasis: Human metastasis-associated mta1 (hMTA1) gene (seq id nos 357-362).
Cell cycle control and signal transduction is strikcly regulated. Frameshift mutations in these genes may result in uncontrolled cell growth. Examples of genes which may be suseptable are: Human protein tyrosine phosphatase (hPTP) gene (seq id nos 95-102), Human kinase (TTK) gene (seq id nos 109-120), Human transcriptional repressor (CTCF) gene (seq id nos 121-127), Human cell cycle regulatory protein (E1A-binding protein) p300 gene (seq id nos 211-218), Human transforming growth factor-beta induced gene product (BIGH3) (seq id nos 227-232), Human FLt4 gene (for transmembrane tyrosinase kinase (seq id nos 280-284), Human G protein-coupled receptor (hGPR1) gene (seq id nos 314-319), Human transcription factor (hITF-2) gene (seq id nos 326-327), Human telomerase-associated protein TP-1 (hTP-1) gene (seq id nos 335-348), Human transcription TFIIB 90 kDa subunit (hTFBIIB90) gene (seq id nos 363-369), Human FADD-homologous ICE/CED-3like protease gene (seq id nos 128-133) 
Mutations in DNA synthesis or -repair enzymes may also lead to uncontrolled cell growth. Human DNA topoisomerase II (top2) gene (seq id nos 103-108) and Human putative mismatch repair/binding protein (hMSH3) gene (seq id nos 134-1471 and (hMSH6) gene (seq id nos 200-203 and 293-297).
The following are tumour suppressor genes, Human retinoblastoma binding protein 1 isoform I (hRBP1) gene (seq id nos 148-156), Human neurofibromin (NF1) gene (seq id nos 176-181), Human p53 associated gene (seq id nos 285-292), Human retinoblastoma related protein (p107) gene (seq id nos 310-313), Human tumour suppressor (hLUCA-1) gene (seq id nos 370-377), Mutations in these genes may result in development of cancer.
The following are oncogenes, proto-oncogenes or putative oncogenes; Human germline n-myc gene (seq id nos 182-188), Human n-myc gene (seq id nos 189-194), Human can (hCAN) gene (seq id nos 298-300), Human dek (hDEK) gene (seq id nos 307-309), b-raf oncogene (seq id nos 170-175), Human DBL (hDBL) proto-cogene/Human MCF2PO (hMCF2PO) gene (seq id nos 301-306). Frameshift mutations in these genes may lead to development of cancer.